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Tuberculosis (TB) is a notifiable zoonotic disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC) that affects a multitude of domestic and wild species. The main lesions caused by these mycobacteria are tuberculous granulomas, which determine the organism's immune response to the disease. Although TB pathogenesis in cattle has been extensively studied, information regarding its progression in other species of interest for the maintenance and transmission of TB such as goats remains limited. This study aimed to characterise the immune response developed in the lungs of goats naturally infected with mycobacteria of MTBC by assessing key cell populations and immunomodulatory molecules involved in defending against TB. Hence, twelve 6-12-month-old Guadarrama kid goats, initially TB-free, were selected and exposed to M. bovis through close contact with other infected goats. Only animals that tested positive by any of the TB diagnostic methods at the end of the experiment were included in the final analysis (n = 9). Gross and microscopic lesions compatible with TB (TBL) in different organs, as well as local response to TB in lungs were evaluated. Our results revealed that after five months of exposure, 44.4 % (4/9) of the M. bovis-infected animals exhibited TBL in the lungs (TBLL+), characterized by a predominance of non-cavitary necrotic granulomas. TBLL+ animals showed significantly higher presence of neutrophils, macrophages (MΦs) and lymphocytes along with greater expression of interferon (IFN)-γ. Conversely, the remaining animals did not present macroscopic or microscopic TBL in the lungs (TBLL-) (5/9). However, these goats displayed elevated expression of toll-like receptors (TLR)2 and TLR4 alongside heightened expression of pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and interleukin (IL)-10. These results suggest the potential development of an effective immune response that may suppress or delay of TBL in infected animals. Further research is needed to elucidate how these molecules, which are involved in the defence against MTBC, confer protection and modulate their expression during infection for TB control., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Two domestic cats (Abyssinian and Carthusian) presented with chronic respiratory signs including cough, respiratory sounds, and polypnea. One of the cats also showed intermittent fever. Thoracic radiographs demonstrated severe changes with predominantly micronodular interstitial lung patterns, some with mineralized areas. Bronchoalveolar lavage detected high-grade pyogranulomatous airway inflammation. A cytological examination for acid-fast bacteria and a PCR for Mycobacteria species of the bronchoalveolar lavage fluid (BALF) were negative in both cats. A culture for Mycobacteria of the BALF detected Mycobacterium avium subspecies in both cats. While the clinical signs, but not the radiographic findings, could be controlled in one cat with prolonged double antibiotic therapy, the other cat developed septic peritonitis shortly after diagnosis and was euthanized. In unexplained chronic respiratory disease in cats, mycobacterial infection should be considered as a differential diagnosis. The breed of the first patient is consistent with the known predisposition for Abyssinians., Competing Interests: Die Autoren bestätigen, dass kein Interessenkonflikt besteht., (Thieme. All rights reserved.)

Mycobacterium microti is a member of the Mycobacterium tuberculosis complex that seldom causes disease in livestock and humans. This study evaluated the effects on immunodiagnosis and the pathological findings in goats after experimental exposure by different routes and doses to M. microti. In a first experiment goats were challenged orally (PO, n = 7) or intranasally (IN, n = 7) with 10 4 CFU. In a second experiment, the endobronchial route was assessed, with a low dose of 10 2 CFU (EB-LD, n = 7) and a high dose of 10 5 CFU (EB-HD, n = 7) as well as the subcutaneous route (SC, n = 5). Temperature, body weight, clinical signs and immunological responses were monitored. Pathological evaluation was carried out and samples were processed for mycobacterial detection. RESULTS: demonstrated the induction of a subclinical pulmonary infection in all the EB-HD challenged animals. Infection was also confirmed in one animal of the SC group, but not in the EB-LD, PO or IN groups. Two animals belonging to the EB-HD and SC groups, respectively, showed positive results to the single intradermal tuberculin test, and another two animals of the EB-HD and EB-LD groups showed doubtful (inconclusive) results, indicating that M. microti can induce mild responses to tuberculin skin testing. No positive results were observed when defined antigens absent in M. microti (ESAT-6 and CPF-10) were used. Our results indicate that animals exposed to M. microti can yield positive results to the skin tests currently performed in livestock tuberculosis eradication campaigns and reinforce the need to use specific antigens in antemortem tests to avoid interference with M. bovis/M. caprae diagnosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic pulmonary aspergillosis (CPA) may mimic pulmonary tuberculosis (PTB). The two diseases are clinically indistinguishable and may result in CPA misdiagnosed as PTB or vice versa. Although PTB is largely recognised as a differential diagnosis of CPA and often ruled out prior to CPA diagnosis, the reverse is uncommon. The aim of this study was to determine the proportion of CPA cases among patients being assessed for PTB. A cross-sectional survey was conducted among consecutive patients referred for GeneXpert Mycobacterium tuberculosis test for the diagnosis of PTB at the Korle-Bu Teaching Hospital, Accra, Ghana. Patients' demographics, clinical and socioeconomic details were obtained using a structured questionnaire. Blood was collected for Aspergillus and HIV serology, and sputum samples obtained for Aspergillus culture. Chest radiograph was obtained, and computed tomography scan was also done for patients with positive Aspergillus serology or cavitation. CPA was defined using an algorithm developed by the Global Action for Fungal Infections (GAFFI) international expert panel. A total of 154 patients were included in the analysis, of whom 134 (87%) did not have a prior PTB diagnosis. There were 41 (26.6%) GeneXpert positive cases. CPA prevalence was 9.7% overall, but 50% in patients with a prior history of PTB and 3.7% in those without previous PTB. Although CPA is rarely considered as a differential diagnosis of PTB in Ghana, our findings show that CPA may affect half of patients being assessed for PTB relapse. Efforts to diagnose CPA should be prioritised in this patient group., (© The Author(s) 2022. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Objective: To determine (1) if chemokine (C-X-C motif) ligand 1 (CXCL1), matrix metalloproteinase 8 (MMP8), interleukin-10 (IL-10), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) can be detected in serum from Asian elephants, and (2) if their concentrations are significantly elevated in Mycobacterium tuberculosis (M.tb) culture-positive elephants compared to -negative elephants. CXCL1, MMP8, IL-10, IFN-γ, and TNF-α were recently identified as potential diagnostic biomarkers for pulmonary tuberculosis in experimental studies in animals and humans. Therefore, we hypothesized that they would be detectable and significantly elevated in M.tb culture-positive elephants compared to M.tb culture-negative elephants., Sample: 101 Asian elephant serum samples, including 91 samples from 6 M.tb-negative elephants and 10 samples from 5 M.tb-positive elephants (none of which exhibited clinical signs of disease). M.tb status was determined by trunk wash culture., Procedures: Commercially available ELISA kits were used to determine the concentrations of each biomarker in serum samples., Results: Biomarker concentrations were below the limit of detection for the assay in 100/101 (99%) samples for CXCL1, 98/101 (97%) samples for MMP8, 85/101 (84%) samples for IL-10, 75/101 (74%) samples for IFN-γ, and 45/101 (45%) samples for TNF-α. Multiple M.tb culture-positive elephants did not have detectable levels of any of the 5 biomarkers., Clinical Relevance: CXCL1, MMP8, IL-10, IFN-γ, and TNF-α were not elevated in M.tb culture-positive Asian elephants compared to M.tb culture-negative Asian elephants. This may be related to disease state (ie, clinically asymptomatic). More sensitive assays are needed to better understand the role of these biomarkers in M.tb infection in Asian elephants.

In 2017, an adult male South American sea lion (Otaria byronia), presenting emaciation and a cervical abscess, stranded alive in Florianópolis, southern Brazil. The animal was directed to a rehabilitation center, dying a few days later. On necropsy, the main gross findings were necrotizing lymphadenitis of the right prescapular lymph node and nodular bronchopneumonia. A novel alphaherpesvirus, tentatively named Otariid alphaherpesvirus 1, was amplified in several tissue samples. No histopathologic findings associated with viral infection were observed. Additionally, pulmonary tuberculosis by Mycobacterium pinnipedii was diagnosed by histopathological, immunohistochemical, and molecular techniques. Several bacteria were cultured from antemortem and postmortem samples, including Proteus mirabilis from the cervical abscess and cardiac blood, and Escherichia coli from the cervical abscess and pericardial effusion. Flavivirus, morbillivirus, and Apicomplexa were not detected by molecular techniques. Herein, we report a novel alphaherpesvirus in a pinniped species of the family Otariidae. Although previously described in Southern Hemisphere pinniped species, including South American sea lions, there is limited information regarding M. pinnipedii impact over this group. Further research is required to determine the associated pathogenesis of this novel herpesvirus, and prevalence of Otariid alphaherpesvirus 1 and M. pinnipedii in the reproductive colonies., (© 2021. Sociedade Brasileira de Microbiologia.)

The Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine is administered parenterally to infants and young children to prevent tuberculosis (TB) infection. However, the protection induced by BCG is highly variable and the vaccine does not prevent pulmonary TB, the most common form of the illness. Until improved TB vaccines are available, it is crucial to use BCG in a manner which ensures optimal vaccine performance. Immunization directly to the respiratory mucosa has been shown to promote greater protection from TB in animal models. γδ T cells play a major role in host defense at mucosal sites and are known to respond robustly to mycobacterial infection. Their positioning in the respiratory mucosa ensures their engagement in the response to aerosolized TB vaccination. However, our understanding of the effect of respiratory BCG vaccination on γδ T cell responses in the lung is unknown. In this study, we used a calf model to investigate the immunogenicity of aerosol BCG vaccination, and the phenotypic profile of peripheral and mucosal γδ T cells responding to vaccination. We observed robust local and systemic M. bovis-specific IFN-γ and IL-17 production by both γδ and CD4 T cells. Importantly, BCG vaccination induced effector and memory cell differentiation of γδ T cells in both the lower airways and peripheral blood, with accumulation of a large proportion of effector memory γδ T cells in both compartments. Our results demonstrate the potential of the neonatal calf model to evaluate TB vaccine candidates that are to be administered via the respiratory tract, and suggest that aerosol immunization is a promising strategy for engaging γδ T cells in vaccine-induced immunity against TB.

Mycobacterium bovis is responsible for bovine tuberculosis in both animals and humans. Despite being one of the most important global zoonotic disease, data related to the ecology and pathogenicity of bovine tuberculosis is scarce, especially in developing countries. In this report, we examined the dynamics of M. bovis transmission among dairy cattle in the Nile Delta of Egypt. Animals belonging to 27 herds from 7 governorates were tested by the Single Intradermal Comparative Skin Tuberculin (SICST), as a preliminary screen for the presence of bovine tuberculosis. Positive SICST reactors were identified in 3% of the animals spread among 40% of the examined herds. Post-mortem examination of slaughtered reactors confirmed the presence of both pulmonary and/or digestive forms of tuberculosis in > 50% of the examined animals. Targeted and whole-genome analysis of M. bovis isolates indicated the emergences of a predominant spoligotype (SB0268) between 2013-2015, suggesting a recent clonal spread of this isolate within the Nile Delta. Surprisingly, 2 isolates belonged to M. bovis BCG group, which are not allowed for animal vaccination in Egypt, while the rest of isolates belonged to the virulent M. bovis clonal complex European 2 present in Latin America and several European countries. Analysis of strain virulence in the murine model of tuberculosis indicated the emergence of a more virulent strain (MBE4) with a specific genotype. More analysis is needed to understand the molecular basis for successful spread of virulent isolates of bovine tuberculosis among animals and to establish genotype/phenotype association.

Red deer (Cervus elaphus) farming is a growing economic activity worldwide. However, the capacity of this species to act as reservoir of animal tuberculosis (TB) poses a threat to other wildlife and to livestock. Diagnostic assay accuracy in this species is therefore highly relevant for prevention and control measures. Our aim was to evaluate the diagnostic performance of the protein complex P22, obtained from Mycobacterium bovis derived purified protein derivative (bPPD), as a candidate antigen for the detection of antibodies against Mycobacterium tuberculosis complex (MTC). We assessed the performance of this new antigen in indirect enzyme-linked immunosorbent assays (ELISA) in TB-positive and TB-negative red deer, in comparison with a bPPD-based ELISA. The P22 ELISA achieved a higher specificity (Sp) and similar sensitivity (Se) in comparison with the bPPD ELISA at all the cut-off points considered. The P22 ELISA yielded optimal Sp (99.02%; 95% confidence intervals [CI 95% ]: 96.5-99.8) and appropriate Se (70.1%; CI 95% : 63.6-76) at the selected cut-off point of 100%. These results suggest that P22 can be used as an alternative antigen in the immunodiagnosis of animal TB through the use of an ELISA-type detection of antibodies against MTC in red deer, thus contributing to the diagnosis of animal TB in this species as a measure for further disease prevention and control programs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) occurs in a wide range of mammalian species and thus poses a health risk to humans living or working in close proximity with TB infected animals. Despite a high incidence of M. bovis infections in domestic or wildlife species tuberculosis infections in rhinoceros have so far been very limited. Over the past 53 years, tuberculosis of the respiratory tract has been confirmed in just 22 rhinoceros, most of those infected not by M. bovis but M. tuberculosis. However, because of the zoonotic risk TB testing is recommended or becomes even mandatory in endangered species. The dilemma in rhinoceros and many other wildlife species; non-validated tests are highly inconsistent in their ability to identify TB infection. Current lack of TB diagnostics may result in TB positive rhinoceros living with the infection, transmitting it to those around them or in euthanasia of animals found unconfirmed at necropsy. This is an unacceptable diagnostic status considering that some species are critically endangered and therefore should not be euthanized in order to confirm suspicion of disease. To overcome this shortcoming we used bronchoscopy to detect mycobacteria in respiratory fluids of TB suspicious rhinoceros. Fluids from seven, TB suspicious white rhinoceros were harvested during 21 bronchoscopies. Our new approach: In addition to bacterial culture a dual quantitative PCR system tested for the general presence of DNA from NTM and more specifically for DNA from MTC. Both, bacterial culture and qPCR were negative for MTC in respiratory fluids of all rhinoceros (7/7). At the same time, respiratory fluids from six rhinoceros tested positive for the presence of NTM or other closely related bacteria (6/7). M. tuberculosis was found only once in an oesophageal aspirate. The high incidence of mycobacterial DNA in the respiratory tract suggests that white rhinoceros, as strict grazers, are immensely exposed to environmental bacteria of this genus. Presence of NTM in the respiratory or intestinal system could possibly cause false positive results in intradermal tests. A wider use of bronchoalveolar lavage is warranted to further elucidate immunologic response to NTM and exposure to, incidence and prevalence of MTC infections in rhinoceros., Competing Interests: Commercial affiliations of authors to Conservation Medicine Services [JC] and the Garston Veterinary Group [DW] do not alter the adherence to all PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.

To screen effective antigens as therapeutic subunit vaccines against Mycobacterium latent infection, we did bioinformatics analysis and literature review to identify effective antigens and evaluated the immunogenicity of five antigens highly expressed in dormant bacteria, which included Rv2031c (HspX), Rv2626c (Hrp1), Rv2007c (FdxA), Rv1738 and Rv3130c. Then, several fusion proteins such as Rv2007c-Rv2626c (F6), Rv2031c-Rv1738-Rv1733c (H83), ESAT6-Rv1738-Rv2626c (LT40), ESAT6-Ag85B-MPT64 <190-198> -Mtb8.4 (EAMM), and EAMM-Rv2626c (LT70) were constructed and their therapeutic effects were evaluated in pulmonary Mycobacterium bovis Bacilli Calmette-Guérin (BCG) - latently infected rabbit or mouse models. The results showed that EAMM and F6 plus H83 had therapeutic effect against BCG latent infection in the rabbit model, respectively, and that the combination of EAMM with F6 plus H83 significantly reduced the bacterial load. In addition, the fusion proteins LT40 and LT70 consisting of multistage antigens showed promising therapeutic effects in the mouse model. We conclude that subunit vaccines consisting of both latency and replicating-associated antigens show promising therapeutic effects in BCG latent infection animal models., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)

Postmortem examination of a wild Asian elephant at Rajiv Gandhi National Park, India, revealed nodular lesions, granulomas with central caseation, and acid-fast bacilli in the lungs. PCR and nucleotide sequencing confirmed the presence of Mycobacterium tuberculosis. This study indicates that wild elephants can harbor M. tuberculosis that can become fatal.

We compared cortisol and thyroid hormone (T3 and T4) concentrations between tuberculosis (TB)-suspected (n=10) and healthy (n=10) elephants of Nepal. Whole blood was collected from captive elephants throughout Nepal, and TB testing was performed using the ElephantTB STAT-PAK ® and DPP VetTB ® serological assays that detect antibodies against Mycobacterium tuberculosis and M. bovis in elephant serum. Cortisol, T3 and T4 were quantified by competitive enzyme immunoassays, and the results showed no significant differences in hormone concentrations between TB-suspect and healthy elephants. These preliminary data suggest neither adrenal nor thyroid function is altered by TB disease status. However, more elephants, including those positively diagnosed for TB by trunk wash cultures, need to be evaluated over time to confirm results.

Background: Tuberculosis caused by Mycobacterium bovis (M. bovis) is very uncommon in horses worldwide., Case Presentation: In the current study, an eight-year-old male Thoroughbred in good body condition was admitted to the Equine Clinic at the Onderstepoort Veterinary Academic Hospital in 2005 due to bilateral epistaxis accompanied by coughing. Routine examinations were conducted to determine the cause of the condition. Endoscopic examination revealed the major source of the epistaxis as the trachea, whereas thoracic radiography indicated the presence of a primary pulmonary mass. M. bovis was isolated from a broncho-alveolar lavage (BAL) sample collected. The pulmonary mass reduced in size three months later following an oral administration of enrofloxacin (7.5 mg/kg PO SID). Genetic fingerprinting by spoligotyping identified the M. bovis isolate as spoligotype SB0868 strain. This M. bovis strain type was never described previously in South Africa (SA). This is the first case of M. bovis infection in a horse in SA which has been fully documented including clinical findings, isolation and genetic characterisation of the causative pathogen., Conclusions: This report indicates that horses may contract and harbour M. bovis despite their lower susceptibility compared to other domestic animals. It also suggests that the infection may be more easily contained and eliminated from the host.

An outbreak of bovine tuberculosis (TB) in a Michigan dairy herd resulted in quarantine, depopulation, pathology, and epidemiologic investigations. This herd, compared to other TB-infected herds in Michigan, was unusual in the long-term feeding of waste milk to its replacement calves. The herd had 80 cattle with positive results on caudal fold test or gamma interferon testing, which were reclassified as suspects because the herd had never been known to be tuberculous previously. Autopsy revealed striking variation in the anatomic distribution of gross anatomic lesions, microscopic lesions, and culture-positive lymph nodes between the adult cattle, the calves, and the domestic cats present on the farm. Adult cattle had lesions and culture-positive lymph nodes predominantly within the thoracic lymph nodes, whereas cats had 50% of their lesions and culture-positive lymph nodes in their abdomens, and 50% of positive calves had culture-positive lymph nodes in their abdomens. This difference in anatomic distribution correlated with the likely routes of infection, which are believed to be by direct airborne transmission in adult cattle and indirect ingestion of contaminated milk in both calves and cats. Although TB literature over the past 100-plus years states that the route of infection may manifest itself in differences in lesion anatomic distribution, our team has been working with TB for over 20 years, and we have never encountered such striking variation between different groups of animals on the same farm., (© 2016 The Author(s).)

Although Mycobacterium tuberculosis infection is an important health concern for Asian elephants (Elephas maximus), no studies have evaluated the associated local immune responses or histologic lesions. In primates including humans, latent tuberculosis is distinguished by well-organized granulomas with TH1 cytokine expression, whereas active disease is characterized by poorly organized inflammation and local imbalance in TH1/TH2 cytokines. This study examined archival, formalin-fixed, paraffin-embedded lung samples from 5 tuberculosis-negative and 9 tuberculosis-positive Asian elephants. Lesions were assessed by light microscopy, and lymphoid infiltrates were characterized by CD3 and CD20 immunolabeling. Expression of TH1 (interferon [IFN]-γ, tumor necrosis factor [TNF]-α) and TH2 (interleukin [IL]-4, IL-10, transforming growth factor [TGF]-β) cytokines was determined using in situ hybridization. In 6 of 9 samples, inflammation was similar to the pattern of primate active disease with low to moderate numbers of lymphocytes, most of which were CD20 positive. In 1 sample, inflammation was most similar to latent tuberculosis in primates with numerous CD3-positive lymphocytes. Expression of IFN-γ was detected in 3 of 8 tuberculosis-positive samples. Expression of TNF-α was detected in 3 of 8 positive samples, including the one with latent morphology. Low-level expression of IL-4 was present in 4 of 8 positive samples. Only single positive samples displayed expression of IL-10 and TGF-β. Tuberculosis-negative samples generally lacked cytokine expression. Results showed heterogeneity in lesions of elephant tuberculosis similar to those of latent and active disease in primates, with variable expression of both TH1 and TH2 cytokines., (© The Author(s) 2014.)

A case of fatal Mycobacterium tuberculosis infection was diagnosed postmortem in a captive 33-yr-old male black rhinoceros (Diceros bicornis) after a nonspecific illness in April 2013. Retrospective testing of sera from this individual revealed that it had been seroreactive by ElephantTB STAT-PAK, dual-path platform VetTB, and multi-antigen print immunoassay for over 12 yr prior to death. Although samples collected at the time of intradermal tuberculin test performed in October 2000 were nonreactive in all three serologic assays, the animal appeared to seroconvert approximately 2.5 wk after the skin test administration. The antibody response remained detectable for the duration of the animal's life (12+ yr), indicating ongoing immunologic stimulation. The current case report supports the use of serologic assays for diagnosis of TB in black rhinoceros and may provide information for earlier detection. However, further research is needed to develop tools for recognition of mycobacterial infections in rhinoceros.

An adult, female, free-ranging red-tailed hawk (Buteo jamaicensis) was presented to a rehabilitation facility for an inability to stand. On examination, it displayed bilateral exaggeration of the pelvic limb reflexes with extensor muscle rigidity, intact superficial pain response, and positive withdrawal reflexes. A complete blood count identified moderate leukocytosis characterized by moderate heterophilia. No abnormalities were appreciable on radiographic evaluation. After initial improvement, it regressed and was euthanized 27 days after presentation. Necropsy and histologic investigation identified reduction in the diameter of the vertebral canal and spinal cord at cervical segments 8-9 with coalescing granulomas and intralesional acid-fast bacilli within the intertrabecular space, left side of the clavicular air sac, and cranial left lung. Bacterial culture and genetic sequencing from respiratory lesions identified Mycobacterium avium avium. Real time-polymerase chain reaction of paraffin-fixed spinal tissue tested positive for M. avium complex. Mycobacteriosis should be considered when peripheral neurologic deficits are present in raptors.

The use of animal models has been invaluable for studying the pathogenesis of Mycobacterium tuberculosis infection, as well as for testing the efficacy of vaccines and drug regimens for tuberculosis. Among the applied animal models, nonhuman primates, particularly macaques, share the greatest anatomical and physiological similarities with humans. As such, macaque models have been used for investigating tuberculosis pathogenesis and preclinical testing of drugs and vaccines. This review focuses on published major studies which illustrate how the rhesus and cynomolgus macaques have enriched and may continue to advance the field of global tuberculosis research., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed.

Mycobacterium bovis causes animal tuberculosis (TB) in cattle, humans, and other mammalian species, including pigs. The goal of this study was to experimentally assess the responses of pigs with and without a history of tonsillectomy to oral vaccination with heat-inactivated M. bovis and challenge with a virulent M. bovis field strain, to compare pig and wild boar responses using the same vaccination model as previously used in the Eurasian wild boar (Sus scrofa), to evaluate the use of several enzyme-linked immunosorbent assays (ELISAs) and lateral flow tests for in vivo TB diagnosis in pigs, and to verify if these tests are influenced by oral vaccination with inactivated M. bovis. At necropsy, the lesion and culture scores were 20% to 43% higher in the controls than those in the vaccinated pigs. Massive M. bovis growth from thoracic tissue samples was observed in 4 out of 9 controls but in none of the 10 vaccinated pigs. No effect of the presence or absence of tonsils was observed on these scores, suggesting that tonsils are not involved in the protective response to this vaccine in pigs. The serum antibody levels increased significantly only after challenge. At necropsy, the estimated sensitivities of the ELISAs and dual path platform (DPP) assays ranged from 89% to 94%. In the oral mucosa, no differences in gene expression were observed in the control group between the pigs with and without tonsils. In the vaccinated group, the mRNA levels for chemokine (C-C motif) receptor 7 (CCR7), interferon beta (IFN-β), and methylmalonyl coenzyme A mutase (MUT) were higher in pigs with tonsils. Complement component 3 mRNA levels in peripheral blood mononuclear cells (PBMC) increased with vaccination and decreased after M. bovis challenge. This information is relevant for pig production in regions that are endemic for M. bovis and for TB vaccine research., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Mycobacterium tuberculosis was cultured from the lung tissues of 3 captive elephants in Nepal that died with extensive lung lesions. Spoligotyping, TbD1 detection and multi-locus variable number of tandem repeat analysis (MLVA) results suggested 3 isolates belonged to a specific lineage of Indo-Oceanic clade, EAI5 SIT 138. One of the elephant isolates had a new synonymous single nucleotide polymorphism (SNP) T231C in the gyrA sequence, and the same SNP was also found in human isolates in Nepal. MLVA results and transfer history of the elephants suggested that 2 of them might be infected with M. tuberculosis from the same source. These findings indicated the source of M. tuberculosis infection of those elephants were local residents, presumably their handlers. Further investigation including detailed genotyping of elephant and human isolates is needed to clarify the infection route and eventually prevent the transmission of tuberculosis to susceptible hosts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Tuberculosis (TB) in non-human primates (NHPs) is highly contagious, requiring efficient identification of animals infected with Mycobacterium tuberculosis. Tuberculin skin test is usually used but lacks desirable sensitivity/specificity and efficiency., Methods: We aimed to develop an immunoassay for plasma antibodies against M. tuberculosis. A key challenge is that not all infected animals contain antibodies against the same M. tuberculosis antigen. Therefore, a multiplex panel of 28 antigens (Luminex(®) -Platform) was developed., Results: Data revealed antibodies against eight antigens (Rv3875, Rv3875-Rv3874 fusion, Rv3874, Rv0934, Rv3881, Rv1886c, Rv2031, Rv3841) in experimentally infected (M. tuberculosis strains: Erdman and H37Rv) NHPs (rhesus and cynomolgus macaques). In a naturally acquired M. tuberculosis infection, rhesus macaques (n = 15) with lung TB pathology (n = 10) contained antibodies to five additional antigens (Rv0831, Rv2220, Rv0054, Rv1099, and Rv0129c)., Conclusions: Results suggest that this user-friendly and easily implementable multiplex panel, containing 13 M. tuberculosis antigens, may provide a high-throughput alternative for NHP TB screening., (© 2014 The Authors. Journal of Medical Primatology published by John Wiley & Sons Ltd.)

An animal keeper was diagnosed with pulmonary tuberculosis (TB) after bi-annual screening for latent TB infection in zoo employees. In the same period, several bonobos of the zoo were suffering from TB as well. The Mycobacterium tuberculosis strains from both the animal keeper and the bonobos appeared identical. We provide evidence that the animals infected their keeper.

The "One world, one health" initiative emphasizes the need for new strategies to control human and animal tuberculosis (TB) based on their shared interface. A good example would be the development of novel universal vaccines against Mycobacterium tuberculosis complex (MTBC) infection. This study uses the goat model, a natural TB host, to assess the protective effectiveness of a new vaccine candidate in combination with Bacillus Calmette-Guerin (BCG) vaccine. Thirty-three goat kids were divided in three groups: Group 1) vaccinated with BCG (week 0), Group 2) vaccinated with BCG and boosted 8 weeks later with a recombinant adenovirus expressing the MTBC antigens Ag85A, TB10.4, TB9.8 and Acr2 (AdTBF), and Group 3) unvaccinated controls. Later on, an endobronchial challenge with a low dose of M. caprae was performed (week 15). After necropsy (week 28), the pulmonary gross pathology was quantified using high resolution Computed Tomography. Small granulomatous pulmonary lesions (< 0.5 cm diameter) were also evaluated through a comprehensive qualitative histopathological analysis. M. caprae CFU were counted from pulmonary lymph nodes. The AdTBF improved the effects of BCG reducing gross lesion volume and bacterial load, as well as increasing weight gain. The number of Ag85A-specific gamma interferon-producing memory T-cells was identified as a predictor of vaccine efficacy. Specific cellular and humoral responses were measured throughout the 13-week post-challenge period, and correlated with the severity of lesions. Unvaccinated goats exhibited the typical pathological features of active TB in humans and domestic ruminants, while vaccinated goats showed only very small lesions. The data presented in this study indicate that multi-antigenic adenoviral vectored vaccines boosts protection conferred by vaccination with BCG.

Animals lacking the inducible nitric oxide synthase gene (nos2(-/-)) are less susceptible to Mycobacterium avium strain 25291 and lack nitric oxide-mediated immunomodulation of CD4(+) T cells. Here we show that the absence of nos2 results in increased accumulation of neutrophils and both CD4(+) and CD8(+) T cells within the M. avium containing granuloma. Examination of the T-cell phenotype in M. avium infected mice demonstrated that CD4(+)CD44(hi) effector T cells expressing the Th1 transcriptional regulator T-bet (T-bet(+)) were specifically reduced by the presence of nitric oxide. Importantly, the T-bet(+) effector population could be separated into CD69(hi) and CD69(lo) populations, with the CD69(lo) population only able to accumulate during chronic infection within infected nos2(-/-) mice. Transcriptomic comparison between CD4(+)CD44(hi)CD69(hi) and CD4(+)CD44(hi)CD69(lo) populations revealed that CD4(+)CD44(hi)CD69(lo) cells had higher expression of the integrin itgb1/itga4 (VLA-4, CD49d/CD29). Inhibition of Nos2 activity allowed increased accumulation of the CD4(+) CD44(hi)T-bet(+)CD69(lo) population in WT mice as well as increased expression of VLA-4. These data support the hypothesis that effector T cells in mycobacterial granulomata are not a uniform effector population but exist in distinct subsets with differential susceptibility to the regulatory effects of nitric oxide., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tuberculosis is a chronic infection caused by strains of the Mycobacterium tuberculosis complex and occurs in both animal and human populations. The death of a tapir showing purulent material and a hard mass in the lungs at necropsy raised suspicion of a potential disease caused by mycobacteria species in a Brazilian zoo. Later, two other tapirs with similar signs died and were further investigated. Polymerase chain reaction (PCR) from bronco-alveolar lavages was performed, and both animals tested positive for the RD(Rio) strain of M. tuberculosis, which is a recently discovered Latin American-Mediterranean sublineage and the main cause of human tuberculosis in Rio de Janeiro, Brazil. To investigate the possibility of human infection and the source of transmission, all 50 zoo employees underwent tuberculin skin testing; four were reactive, but radiographic exams and direct sample staining did not suggest tuberculosis. Thus, direct human to animal transmission was not proven. However, the presence of RD(Rio) M. tuberculosis in tapirs highlights the lack of attention to diseases that human beings may transmit to wildlife.

A female Asian elephant (Elephas maximus) developed vaginal and trunk discharge. Cultures were positive for pan-susceptible Mycobacterium tuberculosis. Isoniazid and pyrazinamide were given rectally and monitored by serum levels. After being trained at 10 mo to accept oral dosing, treatment was changed and rifampin was added. Oral medications were administered for another 10 mo. A year after completion of therapy, the vaginal discharge increased and cultures yielded M. tuberculosis, resistant to isoniazid and rifampin. Treatment with oral ethambutol, pyrazinamide, and enrofloxacin and intramuscular amikacin was initiated. Although followup cultures became negative, adverse reactions to medications precluded treatment completion. Due to public health concerns related to multidrug resistant M. tuberculosis (MDR-TB), the elephant was euthanized. Postmortem smears from the lung, peribronchial, and abdominal lymph nodes yielded acid-fast bacteria, although cultures were negative. This case highlights important considerations in the treatment of M. tuberculosis in animals and the need for a consistent approach to diagnosis, treatment, and follow-up.

The interferon-gamma (IFN-γ) assay is an effective tool for the diagnosis of tuberculosis (Tb) in goats. The objectives of this study were to evaluate factors that might affect assay performance: (1) the phenol concentration of the purified protein derivative (PPD, tuberculin) used; (2) dialysis of PPD; and (3) delaying antigenic stimulation of blood samples for 8, 16 and 24h after collection. The assay was performed in duplicate with two cut-off points. Dialysis of PPD reduced test sensitivity, whereas the concentration of phenol did not significantly affect test outcome. Delaying antigenic stimulation of samples >8h resulted in a reduction in test sensitivity, compromising the capacity of the assay to detect infected animals. Performing the assay in duplicate was unnecessary, which has implications for reducing assay costs. These findings will facilitate the effective application of the IFN-γ assay as an ancillary test in Tb eradication programmes in goats., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Caprine tuberculosis is caused by bacteria of the Mycobacterium tuberculosis complex (Mycobacterium bovis and Mycobacterium caprae). Although typical tuberculoid granulomata are usually observed in the lungs and lymph nodes of infected goats, the presence of cavitary lesions with exuberant mycobacterial growth is also a common feature in this species. The aim of this study was to characterize the immunological mechanisms that lead to liquefaction and cavity formation by comparing granulomata and cavitary lesions. Samples from animals positive by skin testing were collected for microscopical and immunohistochemical examination. Samples were also collected for analysis of cytokine gene expression in the lesions by real time polymerase chain reaction. There were marked differences between granulomata and cavitary lesions. In cavitary lesions there was a substantial population of neutrophils and a significant decrease in the number of CD4(+) T cells, with concomitant increases in other T-cell populations (CD8(+) and cells expressing the γδ form of the T-cell receptor). The enzyme iNOS was strongly expressed by macrophages in the cavitary lesions. There was no difference in the balance of pro- and anti-inflammatory cytokine gene expression in the lesions. These findings suggest that cavitary lesions are reactivation sites, where conditions are optimal for Mycobacterium proliferation and that immunological mechanisms may underlie the severe destruction of lung tissue that characterizes the cavitary pathology., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Mycobacterium africanum consists of two phylogenetically distinct lineages within the Mycobacterium tuberculosis complex, known as M. africanum West African 1 and M. africanum West African 2. These lineages are restricted to West Africa, where they cause up to half of human pulmonary tuberculosis. In this review we discuss the definition of M. africanum, describe the prevalence and restricted geographical distribution of M. africanum West African 1 and 2, review the occurrence of M. africanum in animals, and summarize the phenotypic differences described thus far between M. africanum and M. tuberculosis sensu stricto.

Mycobacterium bovis is a zoonotic member of the Mycobacterium tuberculosis complex responsible for a clinical syndrome indistinguishable from that due to M. tuberculosis. In Australia, infection with M. bovis has historically been associated with employment in the livestock industry or immigration from countries in which animal disease is endemic. It currently accounts for 0.2% of all human cases of tuberculosis within Australia. This paper describes a case of pulmonary M. bovis in a butcher and reviews factors responsible for the declining incidence of this disease in Australia.

Patients with chronic granulomatous disease (CGD), an inherited disorder of phagocytic cells, often contract recurrent life-threatening bacterial and fungal infections. CGD is considered to arise from a functional defect of the O(2)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. To determine whether or not NADPH oxidase is crucial to the host defence against Mycobacterium avium, we investigated the response against M. avium using CGD model mice (gp91-phox(-)) of C57BL/6 strain. A tracheal injection of 1 x 10(7) colony-forming units (CFU)/head of M. avium strain FN into the CGD mice resulted in a pulmonary infection, while also increasing the mortality rate. In contrast, normal C57BL/6 mice injected with same dose of the organisms did not develop severe pulmonary infection and were able to survive through 2 months of observation. The macrophages obtained from the CGD mice were observed to have a higher burden of the bacterial growth than macrophages from normal C57BL/6 mice. These results suggest that the defect of the NADPH oxidase function impairs the host defence against M. avium infection.

Background: This report describes an airborne Mycobacterium avium (MA)-infection in two red-shanked douc langurs (Pygathrix nemaeus nemaeus) from Cologne zoo., Methods: The two individuals and their tissues were investigated clinically (including x-rays), in pathology, in pathohistology, in classical bacteriology and by polymerase chain reaction (PCR)., Results: Clinically, one individual displayed emaciation and a positive reaction in an intrapalpebral testing for M. bovis/MA. The other individual was without any symptoms and did not show any reaction in the intrapalpebral test. In x-ray photos of the lungs, calcified nodules were detected. In pathology, calcified and necrotic nodules were observed within the lungs and the bronchial lymph nodes. In pathohistology, both classical tuberculous granulomas, and few acid fast rods were seen in Ziehl-Neelsen-stain. However, classical bacteriology could not demonstrate mycobacteria. In PCR, MA-infection could be confirmed in one individual using the bronchial lymph nodes., Conclusions: It was an airborne infection; however, the definite source of infection in these cases remained unclear. Animals in contact to the langurs (house sparrows and mice) as well as water used in the building are the most promising candidates. The risk for a zoonotic transmission in these cases has been calculated to be low.

In New Zealand, possums (Trichosurus vulpecula) are the main wildlife reservoir for bovine tuberculosis (Tb), which they transmit to livestock. This study investigated oral vaccination with lipid-formulated Mycobacterium bovis BCG and subsequent protection against virulent M. bovis challenge in wild-caught possums. Possums were trapped from the field and either hand-vaccinated and released back into the wild, or acclimatised to captive conditions prior to voluntary uptake of flavoured vaccine. Possums were subsequently exposed to pulmonary challenge with virulent M. bovis, administered either by instillation of a liquid suspension as an intra-tracheal challenge (field animals) or in micro-droplets as an aerosol (captive animals). Field studies indicated that the relative risk of death in wild possums due to Tb was 2.4 times greater in control compared with orally-vaccinated possums, with the vaccine conferring protection to possums in both good and poor body condition. Laboratory studies indicated that oral vaccination conferred protection in cage-acclimatised possums, with >3log(10) reduction in lung bacterial burdens among vaccinated animals. This study provides evidence that lipid-formulated BCG oral vaccine can provide significant protection to possums in field as well as laboratory conditions, which may favour the use of this formulation as a delivery method for controlling wildlife Tb.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a serious animal health problem in the UK, despite longstanding statutory surveillance and control measures. Endemic infection in the Eurasian badger population is thought to complicate bTB eradication efforts. Sporadic cases of M bovis infection have also been reported in domestic animals other than cattle. Human M bovis infection is extremely rare in the native UK population in the absence of unpasteurised milk consumption or residence abroad. Here, pulmonary TB infection in a UK born female and her pet dog is described, caused by an identical strain of M bovis. Latent TB infection was also identified in a household contact. The potential routes of infection and implications of this case are discussed.

The Mycobacterium bovis strain, bacille Calmette-Guérin (BCG) is one of the most widely used human vaccines and remains one of the safest vaccines available. It has been used in human populations for over 80 years and 100 million children receive the vaccine annually. It has also been employed extensively for vaccine studies in laboratory animal hosts and is currently being developed for use in a variety of livestock and wild animals. Despite the large number of doses delivered since is first usage in 1921, reports of adverse reactions arising from the use of the BCG vaccine are relatively uncommon and where serious reactions do occur they are often the result of vaccination of immuno-compromised individuals. Factors that may influence the development of adverse reactions to BCG include the potency and dose of the vaccine strain, the route of delivery, the age and immune status of the host, and the skill levels of the operator administering the vaccine. Circumstances affecting the notification of adverse reactions include the lack of clear case definitions of abnormal vaccine reactions, and a scarcity of systematic surveillance and functioning reporting systems. With continued use of the BCG and the development of a new generation of prophylactic and therapeutic vaccines against tuberculosis in different host species, the risk factors associated with adverse reactions may need to be reappraised.

We report a case of extensive necrogranulomatous pneumonia due to infection with the dassie bacillus (Mycobacterium tuberculosis complex sp.) in a free-living pregnant adult female dassie (rock hyrax-Procavia capensis). A juvenile female dassie from the same colony also showed a focal lesion in the lungs suggestive of mycobacterial pneumonia. Our findings indicate the widespread occurrence of the dassie bacillus in free-living dassies and suggest very high infection rates in some populations. The introduction of South African dassies into novel environments should be considered in this light.

Mycobacterium tuberculosis (Mtb) is thought to reside in macrophages, although infected dendritic cells (DCs) have been observed. Thus, although cellular associations have been made, global characterization of the cells harboring Mtb is lacking. We have performed temporal and quantitative characterization of the cells harboring Mtb following aerosol infection of mice by using GFP-expressing bacteria and flow cytometry. We discovered that Mtb infects phagocytic cells of diverse phenotypes, that the predominant infected cell populations change with time, and that myeloid DCs are the major cell population infected with Mtb in the lungs and lymph nodes. We also found that the bacteria in the lung-draining lymph node are transported there from the lungs by a CCL19/21-dependent mechanism and that the transport of bacteria to the lymph node is a transient phenomenon despite chronic infection. In addition, we found that the lymph node cell subsets that are most efficacious in stimulating Mtb-specific, TCR-transgenic CD4(+) T lymphocytes are not infected with the bacteria and are scarce or absent from the lungs of infected mice. Finally, we found that the lung cell populations that are infected with Mtb at high frequency are relatively ineffective at stimulating Ag-specific CD4(+) T lymphocytes, and we have obtained evidence that live Mtb can inhibit MHC class II Ag presentation without a decrease in the surface expression of MHC class II. These results indicate that Mtb targets DC migration and Ag presentation in vivo to promote persistent infection.

Pulmonary tuberculosis in guinea pigs is an extremely useful model for drug and vaccine testing due to the fact that its pathological disease process is similar to that present in humans. Progress in this field has been hindered because the tools necessary to undertake a complete immunological analysis of the guinea pig cellular immune response against Mycobacterium tuberculosis have been lacking. In this study, we combined a new flow cytometric gating strategy with immunohistochemistry to track T cells, B cells, and the MIL4 Ab, which detects both guinea pig heterophils (neutrophils) and eosinophils, to provide the first documentation of the kinetics of influx and positioning of these cell populations. The results show that the responding T cells are mostly CD4 cells and that after day 30 of the infection numbers of these cells in the lungs drops dramatically. These appear to be replaced by a steady increase in B cells and granulocytes which was associated with worsening lung pathology. These data reveal new information about the cellular phenotypes which mediate protective immunity or host immunopathogenesis during M. tuberculosis infection in this key animal model.

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-gamma production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that gammadelta T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-gamma in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.

Mycobacterium bovis bacille Calmette Guerin (BCG), the current vaccine against infection with Mycobacterium tuberculosis, offers a variable, protective efficacy in man. It has been suggested that exposure to environmental mycobacteria can interfere with the generation of BCG-specific immunity. We hypothesized that exposure to environmental mycobacteria following BCG vaccination would interfere with established BCG immunity and reduce protective efficacy, thus modeling the guidelines for BCG vaccination within the first year of life. Mice were vaccinated with BCG and subsequently given repeated oral doses of live Mycobacterium avium to model exposure to environmental mycobacteria. The protective efficacy of BCG with and without subsequent exposure to M. avium was determined following an aerogenic challenge with M. tuberculosis. Exposure of BCG-vaccinated mice to M. avium led to a persistent increase in the number of activated T cells within the brachial lymph nodes but similar T cell activation profiles in the lungs following infection with M. tuberculosis. The capacity of BCG-vaccinated mice to reduce the bacterial load following infection with M. tuberculosis was impaired in mice that had been exposed to M. avium. Our data suggest that exposure to environmental mycobacteria can negatively impact the protection afforded by BCG. These findings are relevant for the development of a vaccine administered in regions with elevated levels of environmental mycobacteria.

Little is known regarding the early events of infection of humans with Mycobacterium tuberculosis. The cynomolgus macaque is a useful model of tuberculosis, with strong similarities to human tuberculosis. In this study, eight cynomolgus macaques were infected bronchoscopically with low-dose M. tuberculosis; clinical, immunologic, microbiologic, and pathologic events were assessed 3 to 6 weeks postinfection. Gross pathological abnormalities were observed as early as 3 weeks, including Ghon complex formation by 5 weeks postinfection. Caseous granulomas were observed in the lung as early as 4 weeks postinfection. Only caseous granulomas were observed in the lungs at these early time points, reflecting a rigorous initial response. T-cell activation (CD29 and CD69) and chemokine receptor (CXCR3 and CCR5) expression appeared localized to different anatomic sites. Activation markers were increased on cells from airways and only at modest levels on cells in peripheral blood. The priming of mycobacterium-specific T cells, characterized by the production of gamma interferon occurred slowly, with responses seen only after 4 weeks of infection. These responses were observed from T lymphocytes in blood, airways, and hilar lymph node, with responses predominantly localized to the site of infection. From these studies, we conclude that immune responses to M. tuberculosis are relatively slow in the local and peripheral compartments and that necrosis occurs surprisingly quickly during granuloma formation.